Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
  • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/112—Disease subtyping, staging or classification
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/156—Polymorphic or mutational markers
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/90—Enzymes; Proenzymes
  • G01N2333/914—Hydrolases (3)
  • G01N2333/916—Hydrolases (3) acting on ester bonds (3.1), e.g. phosphatases (3.1.3), phospholipases C or phospholipases D (3.1.4)
  • G01N2333/918—Carboxylic ester hydrolases (3.1.1)
  • G01N2333/92—Triglyceride splitting, e.g. by means of lipase
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/16—Ophthalmology
  • G01N2800/164—Retinal disorders, e.g. retinopathy

Definitions

• Age-related macular degeneration is the most common geriatric eye disorder leading to blindness. Macular degeneration is responsible for visual handicap in what is estimated conservatively to be approximately 16 million individuals worldwide. Among the elderly, the overall prevalence is estimated between 5.7% and 30% depending on the definition of early AMD, and its differentiation from features of normal aging, a distinction that remains poorly understood. Histopathologically, the hallmark of early neovascular AMD is accumulation of extracellular drusen and basal laminar deposit (abnormal material located between the plasma membrane and basal lamina of the retinal pigment epithelium) and basal linear deposit (material located between the basal lamina of the retinal pigment epithelium and the inner collageneous zone of Bruch's membrane).
• the end stage of AMD is characterized by a complete degeneration of the neurosensory retina and of the underlying retinal pigment epithelium in the macular area.
• Advanced stages of AMD can be subdivided into geographic atrophy and exudative AMD.
• Geographic atrophy is characterized by progressive atrophy of the retinal pigment epithelium, hi exudative AMD the key phenomenon is the occurrence of choroidal neovascularisation (CNV).
• CNV choroidal neovascularisation
• Eyes with CNV have varying degrees of reduced visual acuity, depending on location, size, type and age of the neovascular lesion.
• the development of choroidal neovascular membranes can be considered a late complication in the natural course of the disease possibly due to tissue disruption (Bruch's membrane) and decompensation of the underlying longstanding processes of AMD.
• the present invention is directed to methods and compositions that allow for improved diagnosis of AMD and susceptibility to AMD.
• the compositions and methods of the invention are directed to the discovery of genetic markers associated
• the present invention is directed toward a method for diagnosing AMD or a susceptibility to AMD, a protective phenotype for AMD, or a neutral genotype for AMD, comprising detecting the presence or absence of a particular allele at a polymorphic site associated with lipid metabolism genes, where the allele is indicative of AMD or a susceptibility to AMD.
• the polymorphic site is a single nucleotide polymorphism associated with high density lipoprotein cholesterol (HDL-c) pathway genes.
• the polymorphic site is rs493258 (SEQ ID NO:1), where the cytidine polymorphism is indicative of AMD or susceptibility to AMD.
• the polymorphic site is selected from the group
• rsl 1755724 (SEQ ID NO:2), wherein the adenine polymorphism is indicative of AMD or susceptibility to AMD
• rsl 3095226 (SEQ ID NO:3), wherein the cytidine polymorphism is indicative of AMD or susceptibility to AMD
• rsl 931897 (SEQ ID NO:4), wherein the cytidine polymorphism is indicative of AMD or susceptibility to AMD
• rs509859 (SEQ ID NO:5), wherein the thymine
• rs7626245 SEQ ID NO:6
• rs3748391 SEQ ID NO: 7
• guanine polymorphism is indicative of AMD or susceptibility to AMD
• rs4628134 SEQ ID NO:8
• thymine polymorphism is indicative of AMD or susceptibility to AMD
• rs2059883 SEQ ID NO: 10
• thymine polymorphism is indicative of AMD or susceptibility to AMD
• rsl 95484 SEQ ID NO:11
• cytidine polymorphism is indicative of AMD or susceptibility to AMD
• rsl 0739343 SEQ ID NO: 12
• adenine polymorphism is indicative of
• rsl 7628762 (SEQ ID NO: 13), wherein the adenine polymorphism is indicative of AMD or susceptibility to AMD
• rs4883193 (SEQ ID NO: 14), wherein the adenine polymorphism is indicative of AMD or susceptibility
• the guanine polymo ⁇ hism is indicative of AMD or susceptibility to AMD; rsl457239 (SEQ ID NO:22), wherein the guanine polymo ⁇ hism is indicative of AMD or susceptibility to AMD; rsl 0089310 (SEQ ID NO:23), wherein the adenine polymo ⁇ hism is indicative of AMD or susceptibility to AMD; rs2803544 (SEQ ID NO:24), wherein the adenine polymo ⁇ hism is indicative of AMD or susceptibility
• rs3019878 SEQ ID NO:25
• rsl6908453 SEQ ID NO:26
• rsl 3080329 SEQ ID NO:27
• thymine polymo ⁇ hism is indicative of AMD or susceptibility to AMD
• rs2842895 SEQ ID NO:28
• polymo ⁇ hism is indicative of AMD or susceptibility to AMD; rs 1511454 (SEQ ID NO:29), wherein the guanine polymo ⁇ hism is indicative of AMD or susceptibility to AMD; rsl 468678 (SEQ ID NO:30), wherein the adenine polymo ⁇ hism is indicative of AMD or susceptibility to AMD; rs887656 (SEQ ID NO:31), wherein the thymine polymo ⁇ hism is indicative of AMD or susceptibility to AMD;
• rsl 403264 (SEQ ID NO:32), wherein the cytidine polymo ⁇ hism is indicative of AMD or susceptibility to AMD
• rslO812380 (SEQ ID NO:33)
• the guanine polymo ⁇ hism is indicative of AMD or susceptibility to AMD
• rs6933716 (SEQ ID NO:34)
• the thymine polymo ⁇ hism is indicative of AMD or susceptibility to AMD
• rs2290465 (SEQ ID NO:35), wherein the guanine polymo ⁇ hism is
• rsl7762454 SEQ ID NO:36
• thymine polymo ⁇ hism is indicative of AMD or susceptibility to AMD
• rsl 333049 SEQ ID NO:37
• the present invention is directed toward a method for determining AMD risk in a patient, including: obtaining a patient sample, detecting 5 an AMD marker in the patient sample further comprising determining the presence or absence of a particular allele at a polymorphic site associated with one or more metalloproteinase genes, wherein the allele indicates: a susceptibility for AMD, a protective phenotype for AMD or a neutral genotype for AMD, thereby indicating AMD risk in the patient.
• the allele at a polymorphic site is a
• the allele at a polymorphic site is a single nucleotide polymorphism associated with a TIMP3 gene.
• the allele includes SEQ ID NO.39 and a cytidine polymorphism within the allele is indicative of susceptibility to AMD or increased pathogenesis of AMD in the patient.
• the presence or absence of a particular allele is detected by a hybridization assay. In another embodiment, the presence or absence of a particular allele is determined using a microarray. In yet another embodiment, the presence or absence of a particular allele is determined using an antibody. In one aspect, the present invention is directed toward a method for
• identifying a subject who is at risk or protected from developing AMD including detecting the presence or absence of at least one at risk allele at rs493258 and one at risk allele at rsl0468017; and detecting the presence or absence of at least one at risk allele or protective allele associated with LIPC gene; where a subject is not at risk if the subject is one of about 20% of the population with a less than about 1 % risk of
• the subject is at risk if the subject is one of about 1% of the population with a greater than about 50% risk of developing AMD.
• the present invention is directed toward a method for identifying a subject who is at risk or protected from developing AMD, including detecting the presence or absence of at least one at risk allele at rs9621532; and
• BOST 1508298.1 the subject is at risk if the subject is one of about 1% of the population with a greater than about 50% risk of developing AMD.
• the presence or absence of a particular allele is detected by a hybridization assay. In another embodiment, the presence or absence of a 5 particular allele is determined using a microarray.
• the present invention is directed toward a purified polynucleotide comprising the polymorphic site and at least about six or more contiguous nucleotides of one or more of the sequences given as SEQ ID NOS: 1-39, where the variant allele is present at the polymorphic site.
• the present invention is directed toward a diagnostic array comprising one or more polynucleotide probes that are complementary to a polynucleotide of SEQ ID NOS: 1-39.
• the present invention is directed toward a diagnostic system including: a diagnostic array, an array reader, an image processor, a database having 15 data records and information records, a processor, and an information output; wherein the system compiles and processes patient data and outputs information relating to the statistical probability of the patient developing AMD.
• the present invention is directed toward a method of using the diagnostic system, including contacting a subject sample to the diagnostic array 20 under high stringency hybridization conditions; inputting patient information into the system; and obtaining from the system information relating to the statistical probability of the patient developing AMD.
• the present invention is directed toward a method of making the diagnostic array, including: applying to a substrate at a plurality 25 particular address on the substrate a sample of the individual purified polynucleotide compositions comprising SEQ ID NOS: 1-39.
• the present invention is directed toward a method for diagnosing AMD or a susceptibility to AMD in a subject comprising combining genetic risk with behavioral risk, wherein the genetic risk is determined by detecting 30 the presence or absence of a particular allele at a polymorphic site associated with a lipid metabolism or metalloproteinase genes, wherein the allele is indicative of AMD or a susceptibility to AMD.
• the polymorphic site is rs493258 (SEQ ID NO: 1), where the cytidine polymorphism is indicative of AMD or susceptibility to AMD.
• the presence or absence of a particular allele is detected by a hybridization assay. In another embodiment, the presence or absence of a 5 particular allele is determined using a microarray. In still another embodiment, the presence or absence of a particular allele is determined using an antibody.
• the behavioral risk is assessed by determining if the subject exhibits a behavior or trait selected from the group consisting of: obesity, smoking, vitamin and dietary supplement intake, use of alcohol or drugs, poor diet 10 and a sedentary lifestyle.
• the elevated BMI is used to determine obesity.
• FIG. 1 A is a graph showing the distribution of effects from the discovery phase on analysis of 726,970 SNPs.
• the points coded in blue are loci from the 15 previous AMD associated regions including CFH, ARMS2, CFI, C2, and C3.
• FIG. I B is a graph showing the distribution of P-values with the previously reported AMD associations removed, resulting in exclusion of 159 SNPs from this figure.
• the lambda of this distribution is approximately 1.04.
• FIG. 2A is a graph showing the first two principle components for GWAS of 20 AMD and MIGEN. The AMD samples are in red and the controls are in blue.
• FIG. 2B is a graph showing the first two axes of variation for requiring 99% call rate for all SNPs.
• the AMD samples are in red and the MIGEN controls are in blue.
• the samples with a value greater than 0.02 on the second component have been excluded.
• FIG. 3 is a graph showing 80% power to detect a biallelic CNV (copy number variation). Based on the sample size used in the CNV analysis of 459 controls and 838 cases the power was plotted to detect a bialleleic CNV (for example a simple deletion) at the pO.OOl level.
• FIG. LIPC sequence analysis and retinal expression.
• BOST 1508298.1 Protein analysis of adult bovine retinal LIPC expression. Undetectable levels of LIPC from 150 micrograms of bovine retinal extract prompted an immunoprecipitation (IP) experiment from 5 milligrams of bovine retinal lysate. Specificity of the 55 kDa LIPC band from the LIPC IP was validated using a
• FIG. Immunoblot of hepatic lipase antibody (H-70, SC-21007) against
• Lane 1 human whole protein extracts. Lane 1, macular retina; Lane 2, peripheral retina; Lane 3, RPEchoroid; Lane 4, liver. Lanes 1-3 were normalized against actin.
• FIG. 6 Distribution of hepatic lipase C (red) in central (A) and peripheral (B) monkey retina using a rabbit polyclonal antibody (Cat. #SC-21007) raised against amino acids 91-160 of human origin. This antibody labels all retina neurons,
• ONL outer nuclear layer
• INL inner nuclear layer
• IPL inner plexiform layer
• GCL ganglion cell layer.
• FIG. 7 are sequences showing alleles at polymorphic sites: rs493258 (SEQ ID NO:1), rsl 1755724 (SEQ ID N0:2), rs 13095226 (SEQ ID NO:3), rsl931897 (SEQ ID NO:4), rs509859 (SEQ ID N0:5), rs7626245 (SEQ ID NO:6), rs3748391 (SEQ ID N0:7), rs4628134 (SEQ ID N0:8), rsl2637095 (SEQ ID NO:9), 5 rs2059883 (SEQ ID NO:10), rsl95484 (SEQ ID NO:11), rsl 0739343 (SEQ ID NO:
• the present invention is directed to the discovery that particular alleles at polymorphic sites associated with genes coding for proteins associated with lipid
• hepatic lipase gene in the high-density lipoprotein cholesterol (HDL-c) pathway
• HDL-c high-density lipoprotein cholesterol
• gene is a term used to describe a genetic element that gives rise to expression products (e.g., pre-mRNA, mRNA and polypeptides).
• 25 includes regulatory elements, exons and sequences that otherwise appear to have only structural features, e.g., introns and untranslated regions.
• the genetic markers are particular "alleles” at "polymorphic sites” associated with the hepatic lipase gene (LIPC).
• a nucleotide position at which more than one nucleotide can be present in a population either a natural population or a synthetic
• polymorphic site Where a polymorphic site is a single nucleotide in length, the site is referred to as a single nucleotide polymorphism ("SNP"). If at a particular
• BOST 1508298.1 chromosomal location for example, one member of a population has an adenine and another member of the population has a thymine at the same genomic position, then this position is a polymorphic site, and, more specifically, the polymorphic site is a SNP.
• Polymorphic sites can allow for differences in sequences based on 5 substitutions, insertions or deletions. Each version of the sequence with respect to the polymorphic site is referred to herein as an "allele" of the polymorphic site.
• the SNP allows for both an adenine allele and a thymine allele.
• a genetic marker is "associated" with a genetic element or phenotypic trait
• association indicates physical association, e.g. , proximity in the genome or presence in a haplotype block, of a marker and a genetic element.
• a reference sequence is typically referred to for a particular genetic element, e.g., a gene. Alleles that differ from the reference are referred to as “variant” alleles.
• the reference sequence often chosen as the most frequently occurring allele or as the allele conferring a typical phenotype, is referred to as the "wild-type" allele.
• Some variant alleles can include changes that affect a polypeptide, e.g., the
• sequence differences when compared to a reference nucleotide sequence, can include the insertion or deletion of a single nucleotide, or of more than one nucleotide, resulting in a frame shift; the change of at least one nucleotide, resulting in a change in the encoded amino acid; the change of at least one nucleotide, resulting in the generation of a
• nucleotide sequence 25 premature stop codon; the deletion of several nucleotides, resulting in a deletion of one or more amino acids encoded by the nucleotides; the insertion of one or several nucleotides, such as by unequal recombination or gene conversion, resulting in an interruption of the coding sequence of a reading frame; duplication of all or a part of a sequence; transposition; or a rearrangement of a nucleotide sequence.
• AMD can be a synonymous change in one or more nucleotides (i.e., a change that does not result in a change to a codon of a complement pathway gene).
• BOST_1508298.1 polymorphism can, for example, alter splice sites, affect the stability or transport of mRNA, or otherwise affect the transcription or translation of the polypeptide.
• the polypeptide encoded by the reference nucleotide sequence is the "reference” polypeptide with a particular reference amino acid sequence, and polypeptides 5 encoded by variant alleles are referred to as "variant" polypeptides with variant amino acid sequences.
• Haplotypes are a combination of genetic markers, e.g., particular alleles at polymorphic sites.
• the haplotypes described herein are associated with AMD and/or a susceptibility to AMD. Detection of the presence or absence of the haplotypes described herein.
• haplotypes herein therefore is indicative of AMD, a susceptibility to AMD or a lack thereof.
• the haplotypes described herein are a combination of genetic markers, e.g. , SNPs and microsatellites. Detecting haplotypes, therefore, can be accomplished by methods known in the art for detecting sequences at polymorphic sites.
• haplotypes and markers disclosed herein are in "linkage disequilibrium"
• LD hepatic lipase gene
• HDL-c high-density lipoprotein cholesterol pathway
• Linkage refers to a higher than expected statistical association of genotypes and/or phenotypes with each other.
• LD refers to a non-random assortment of two genetic elements. If a particular genetic element
• the elements 20 e.g., an allele at a polymorphic site
• the predicted occurrence of a person's having both elements is 0.125, assuming a random distribution of the elements. If, however, it is discovered that the two elements occur together at a frequency higher than 0.125, then the elements are said to be in
• Allele frequencies can be determined in a population, for example, by genotyping individuals in a population and determining the occurrence of each allele in the
• populations of diploid individuals e.g., human populations
• individuals will typically have two alleles for each genetic element (e.g., a marker or gene).
• the invention is also directed to markers identified in a "haplotype block” or "LD block". These blocks are defined either by their physical proximity to a genetic element, e.g. , a lipoprotein pathway gene, or by their "genetic distance” from the element. Markers and haplotypes identified in these blocks, because of their 5 association with AMD and the lipoprotein metabolic pathways, are encompassed by the invention.
• a genetic element e.g. , a lipoprotein pathway gene
• Markers and haplotypes identified in these blocks because of their 5 association with AMD and the lipoprotein metabolic pathways, are encompassed by the invention.
• identification of a marker associated with a phenotype identifies the block as associated with the phenotype. Any marker identified within the block can therefore be used to indicate the phenotype.
• surrogate markers 15 haplotypes are referred to herein as "surrogate" markers.
• Such a surrogate is a marker for another marker or another surrogate marker.
• Surrogate markers are themselves markers and are indicative of the presence of another marker, which is in turn indicative of either another marker or an associated phenotype.
• Susceptibility for developing AMD includes an asymptomatic patient
• Susceptibility for not developing AMD includes an asymptomatic patient, wherein the presence of the wild type allele indicating a lack of predisposition to AMD.
• T1MP3 Tissue Inhibitor of MetalloProteinases-3
• IMPG2 the gene encoding the retinal interphotoreceptor matrix (IPM) proteoglycan IPM 200
• VMD2 the bestrophin gene
• ELO VL4 elongation of very long chain fatty acids
• RDS peripheral blood serum
• EFEMPl EGF-containing fibulin-like extracellular matrix
• BMD bestrophin
• GPR75 a G protein coupled receptor gene. Others have shown a possible association with the disease in at least one study- PONl the (paraoxonase gene); SOD2 (manganese superoxide dismutase; APOE
• BOST_1508298.1 (apolipoprotein E), in which the ⁇ 4 allele has been found to be associated with the disease in some studies and not associated in others; and CST3 (cystatin C), where one study has suggested an increased susceptibility for ARMD in CST3 B/B homozygotes.
• ABCR 5 ABC4
• the invention comprises an array of gene fragments, particularly including those SNPs given as SEQ ID NOS: 1-39, and probes for detecting the allele at the SNPs of SEQ ID NOS: 1-39.
• Polynucleotide arrays io provide a high throughput technique that can assay a large number of polynucleotide sequences in a single sample. This technology can be used, for example, as a diagnostic tool to assess the risk potential of developing AMD using the SNPs and probes of the invention.
• Polynucleotide arrays (for example, DNA or RNA arrays), include regions of usually different sequence polynucleotides arranged in a
• the 20 in the sample can be labeled with a suitable label (such as a fluorescent compound), and the fluorescence pattern on the array accurately observed following exposure to the sample. Assuming that the different sequence polynucleotides were correctly deposited in accordance with the predetermined configuration, then the observed binding pattern will be indicative of the presence and/or concentration of one or
• Arrays can be fabricated by depositing previously obtained biopolymers onto a substrate, or by in situ synthesis methods.
• the substrate can be any supporting material to which polynucleotide probes can be attached, including but not limited to glass, nitrocellulose, silicon, and nylon. Polynucleotides can be bound to the
• GeneChipTM can also be used.
• Use of the GeneChipTM, to detect gene expression is described, for example, in Lockhart et al, Nat. Biotechnol., 14:1675, 1996; Chee et al., Science, 274:610, 1996; Hacia et al, Nat. Gen., 14:441, 1996; and Kozal et al, Nat. Med., 2:753, 1996.
• Other types of arrays are known in the art, and are sufficient for developing an AMD diagnostic array of the present invention.
• single-stranded polynucleotide probes can be spotted onto a substrate in a two-dimensional matrix or array.
• Each single-stranded polynucleotide probe can comprise at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, or 30 or more contiguous nucleotides selected from the nucleotide sequences shown in SEQ ID NO: 1-39, or the complement thereof.
• 20 comprise at least one single-stranded polynucleotide probe comprising at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, or 30 or more contiguous nucleotides selected from the nucleotide sequences shown in SEQ ID NO: 1-39, or the complement thereof.
• Tissue samples from a subject can be treated to form single-stranded
• Detectable labels that can be used include but are not limited to radiolabels, biotinylated labels, fluorophors, and chemiluminescent labels. Double stranded polynucleotides, comprising the
• sample polynucleotides bound to polynucleotide probes can be detected once the unbound portion of the sample is washed away. Detection can be visual or with computer assistance. Preferably, after the array has been exposed to a sample,
• the array is read with a reading apparatus (such as an array "scanner") that detects the signals (such as a fluorescence pattern) from the array features.
• a reading apparatus such as an array "scanner”
• Such a reader preferably would have a very fine resolution (for example, in the range of five to twenty microns) for an array having closely spaced features. 5
• the signal image resulting from reading the array can then be digitally processed to evaluate which regions (pixels) of read data belong to a given feature as well as to calculate the total signal strength associated with each of the features.
• feature extraction U.S. Pat No. 7,206,438, Using any of the feature extraction techniques so
• detection of hybridization of a patient derived polynucleotide sample with one of the AMD markers on the array given as SEQ ID NO: 1-39 identifies that subject as having or not having a genetic risk factor for AMD, as described above.
• the invention provides a system for compiling
• a computer aided medical data exchange system is preferred.
• the system is designed to provide high-quality medical care to a patient by facilitating the management of data available to care providers.
• the care providers will typically include physicians, surgeons, nurses, clinicians, various specialists, and so forth. It should
• the system provides an interface, which allows the clinicians to exchange data with a data processing system.
• the data processing system is linked to an integrated knowledge base and a database.
• the database may be software-based, and includes data access tools for drawing information from the various resources as described below, or coordinating or translating the access of such information.
• the database will unify raw data into a useable form. Any suitable form may be employed, and multiple forms may be employed, where desired, including hypertext markup language (HTML)
• the integrated knowledge base is considered to include any and all types of
• BOST_1508298.1 available medical data that can be processed by the data processing system and made available to the clinicians for providing the desired medical care.
• data within the resources and knowledge base are digitized and stored to make the data available for extraction and analysis by the database and the data processing system. 5
• the data is placed in a form that permits it to be identified and manipulated in the various types of analyses performed by the data processing system.
• the integrated knowledge base is intended to include one or more repositories of medical -related data in a broad sense, as well as interfaces and
• the data itself may relate to patient-specific characteristics as well as to non-patient specific information, as for classes of persons, machines, systems and so forth.
• the repositories may include devoted systems for storing the
• the repositories and processing resources making up the integrated knowledge base may be expandable and may be physically resident at any number of locations, typically linked by dedicated or open network links.
• the data contained in the integrated knowledge base may include both clinical data (e.g.,
• Examples of preferred clinical data include patient medical histories, patient serum, plasma, and/or other biomarkers such as blood levels of certain other nutrients, fats, female and male hormones, etc., and cellular antioxidant levels, and the identification of past or current environmental, lifestyle and other factors that predispose a patient to
• Non-clinical data may include more general information about the patient, such as residential address, data relating to an insurance carrier, and names and addresses or phone numbers of significant or recent
• the flow of information can include a wide range of types and vehicles for information exchange.
• the patient can interface with clinicians through conventional clinical visits, as well as remotely by telephone, electronic mail, forms, and so forth.
• the patient can also interact with elements of the resources via a range 5 of patient data acquisition interfaces that can include conventional patient history forms, interfaces for imaging systems, systems for collecting and analyzing tissue samples, body fluids, and so forth.
• Interaction between the clinicians and the interface can take any suitable form, depending upon the nature of the interface.
• the clinicians can interact with the data processing system through
• the links between the interface, data processing system, the knowledge base, the database and the resources typically include computer data exchange interconnections, network connections, local area networks, wide area networks, dedicated networks, virtual private network, and so
• the resources can be patient-specific or patient-related, that is, collected from direct access either physically or remotely (e.g., via computer link) from a patient.
• the resource data can also be population-specific so as to permit analysis of specific patient risks and conditions based upon comparisons to known
• the resources can generally be thought of as processes for generating data. While many of the systems and resources will themselves contain data, these resources are controllable and can be prescribed to the extent that they can be used to generate data as needed for appropriate treatment of the patient. Exemplary controllable and prescribable
• 25 resources include, for example, a variety of data collection systems designed to detect physiological parameters of patients based upon sensed signals.
• electrical resources can include, for example, electroencephalography resources (EEG), electrocardiography resources (ECG), electromyography resources (EMG), electrical impedance tomography resources (EIT), nerve conduction test resources,
• imaging resources can be controlled and prescribed as indicated at reference numeral.
• a number of modalities of such resources are currently available, such as,
• BOST_1508298.1 for example, X-ray imaging systems, magnetic resonance (MR) imaging systems, computed tomography (CT) imaging systems, positron emission tomography (PET) systems, fluorography systems, sonography systems, infrared imaging systems, nuclear imaging systems, thermoacoustic systems, and so forth.
• Imaging systems 5 can draw information from other imaging systems, electrical resources can interface with imaging systems for direct exchange of information (such as for timing or coordination of image data generation, and so forth).
• Such resources include blood, urine, saliva and other fluid analysis resources, including gastrointestinal, reproductive, urological, nephrological (kidney function), and cerebrospinal fluid analysis system.
• Such resources can further include polymerase (PCR) chain reaction analysis systems, genetic marker analysis systems, radioimmunoassay systems, chromatography and similar chemical analysis systems,
• Histologic resources somewhat similarly, can be included, such as tissue analysis systems, cytology and tissue typing systems and so forth. Other histologic resources can include immunocytochemistry and histopathological analysis systems. Similarly, electron and other microscopy systems, in situ hybridization systems, and so forth can
• Pharmacokinetic resources can include such systems as therapeutic drug monitoring systems, receptor characterization and measurement systems, and so forth. Again, while such data exchange can be thought of passing through the data processing system, direct exchange between the various resources can also be implemented.
• Use of the present system involves a clinician obtaining a patient sample, and evaluation of the presence of a genetic marker in that patient indicating a predisposition (or not) for AMD, such as SEQ ID NO: 1 -39, alone or in combination with other known risk factors.
• the clinician or their assistant also obtains appropriate clinical and non-clinical patient information, and inputs it into the
• the system then compiles and processes the data, and provides output information that includes a risk profile for the patient, of developing AMD.
• the present invention thus provides for certain polynucleotide sequences that have been correlated to AMD. These polynucleotides are useful as diagnostics, and are preferably used to fabricate an array, useful for screening patient samples.
• the array in a currently most preferred embodiment, is used as part of a laboratory 5 information management system, to store and process additional patient information in addition to the patient's genomic profile. As described herein, the system provides an assessment of the patient's risk for developing AMD, risk for disease progression, and likelihood of disease prevention based on patient controllable factors.
• Age-related macular degeneration (AMD), the leading cause of late onset blindness, arises from retinal damage associated with accumulation of drusen and
• BOST_1508298.1 but could represent a pleiotropic effect of the same functional component.
• This genetic locus implicates a different biologic pathway than previously reported and provides a new avenue for possible prevention and treatment of AMD.
• Age-related macular degeneration is a common, late-onset disorder 5 that is modified by covariates including smoking and BMI, and has a 3-6 fold higher recurrence ratio in siblings than in the general population.
• the burden of AMD is clinically significant, causes visual loss, and reduces quality of life.
• individuals age 75 or older approximately one in four have some sign of this disease, while about one in 15 have the advanced form with visual loss.
• Small io genome- wide association studies (GWAS), candidate gene screening, and focused examination of regions identified in linkage studies revealed significant associations between AMD and common genetic variations at CFH loci on chromosome 1 and the ARMS2/HTRA1 region on chromosome 10.
• Samples were genotyped on the Affymetrix 6.0 platform which contains probes for 906,000 SNPs and an additional 946,000 SNP-invariant probes to enhance copy number variation (CNV) analysis and captures 82% of the variation at an r 2 >0.8 for Europeans in the 3.1 million SNPs of ⁇ apMap phase 2.
• CNV copy number variation
• BOST_1508298.1 lipase a gene involved in triglyceride hydrolysis and high density lipoprotein (HDL) function, thus revealing a novel pathway involved in AMD pathogenesis.
• TIMP3 has been found to be a matrix-bound angiogenesis inhibitor and mutations in the gene itself have been shown to induce abnormal neovascularization.
• COL8A1 encodes one of the two alpha chains of type VIII collagen.
• the gene product is a short chain collagen and a major component of the multiple basement membranes in the eye including Bruch's membrane and the choroidal stroma.
• Bruch's membrane is located directly below the retinal pigment epithelium and plays a central role in the pathogenesis of AMD.
• LIPC encoding hepatic triglyceride lipase catalyzes hydrolysis of phospholipids, mono-, di-, triglycerides, acyl-CoA thioesters and is a critical enzyme in HDL metabolism.
• Hepatic lipase also binds heparin and has the dual functions of triglyceride hydrolase and ligand/bridging factor for receptor-mediated lipoprotein uptake.
• LIPC has been shown to be expressed in the retina through our experiments, and CETP and ABCAl have previously been shown to be expressed in the retina.
• Controls were unrelated to cases, 60 years of age or older, and were defined as individuals without macular degeneration, categorized as CARMS stage 1, based
• BOSTJ 508298.1 was comprised of DNA samples from unrelated Caucasian individuals not included in the GWAS, including 868 advanced AMD cases and 410 examined controls who were identified from the same Tufts cohorts, and 379 unexamined MGH controls. Genotyping, Analysis, and Replication.
• the GWAS genotyping and the Tufts/MGH follow-up replication genotyping were performed at the Broad and National Center for Research Resources (NCRR) Center for Genotyping and Analysis using the Affymetrix SNP 6.0 GeneChip and the Sequenom MassARRAY system for iPLEX assays, respectively.
• NCRR National Center for Research Resources
• Affymetrix 6.0 GeneChip which passed quality control filters. Then 43,562 SNPs were removed for low call rate, 4,708 were removed for failing Hardy- Weinberg test at 10-3, and 8,332 SNPs were removed because of failing a differential missing test between cases and controls at 10-3. Finally, 126,050 SNPs were removed for having
• MIGEN Myocardial Infarction Genetics
• Tufts/MGH Affy represents the genome-wide association scan using the Affymetrix 6.0 platform from Tufts Medical Center, Tufts University School of Medicine, without the MIGEN controls included; Tufts/MGH Replication represents the follow up replication pool at MGH/Tufts; UM ILMN represents the genome-wide association scan using the Illumina 322 platform from the University of Michigan; JHU represents the Johns Hopkins University sample replication, and NY represents
• AMD Grading System grade 1 represents individuals with no drusen or a few small drusen, 4 represents individuals with central or non-central geographic atrophy ("advanced dry type"), and 5 represents individuals with neovascular disease ("advanced wet type").
• Each step represents a cleaning stage.
• the initial sample represents all samples genotyped.
• the initial dataset cleaning encompasses HWE, call rate, differential 5 missingness between cases and controls, and minor allele frequency threshold.
• call rate Adding in shared controls, the call rate and MAF thresholds were reapplied. For the final stage, call rate of 99% was required as was the removal of individuals who did not cluster with the majority of the sample.
• Tufts represents the discovery genome-wide scan, without the addition of the MIGEN shared controls, Tufts/MGH includes the Tufts Medical Center, Tufts University School of Medicine sample plus the MIGEN shared controls, MPM refers to
• This table represents the number of loci with a P- value less than the threshold 5 excluding known loci. As we increase the total sample size, the significance for our likely SNP continues to move in the same direction.
• CHISQ The chi-square for each panel versus the other panels as implemented in PLINK.
• DF Degree of Freedom for each test.
• P p-value where the lower numbers represents greater heterogeneity.
• Panel The 15 samples tested in each phase of our meta-analysis.
• Combined P value refers to the combination of the Tufts/MGH, UM, and Replication datasets.
• Tufts represents the discovery genome-wide scan, without the addition of the MIGEN shared controls
• Tufts/MGH includes the Tufts Medical Center, Tufts University School of Medicine sample plus the MIGEN shared controls
• UM refers to the University of Michigan's shared results from the genome-wide association scan
• Meta-analysis refers to the combined P-value for the meta-analysis of Tufts/MGH and UM genome-wide scans.
• SNP single nucleotide polymorphism
• Chr chromosome
• Position base pair position in NCBI 36.1 ; Al : Minor Allele; A2: 5 Major Allele; MAF: Minor Allele Frequency; # Associated SNPs: number of other SNPs in the region showing association in our discovery scan; Tufts/MGH P: P-value of our original GWAS scan; Tufts/MGH Repl P: /"-value of Tufts/MGH replication cohort; MPM P: P- value of Michigan/Penn/Mayo GWAS; Repl Cohort P: P- value of total combined weighted average Z score meta-analysis across the individual replication cohort sites from (a) JHU (Johns Hopkins University), (b) COL (Columbia University), (c) UT (University of Utah), (d) FR-CRET (Hopital Intercommunal de Creteil), and (e) WASH U (Washington University) as denoted; Meta Z 10 score: weighted
• Tufts/MGH-NoMIGEN our discovery GWAS cohort without the MIGEN controls
• Tufts/MGH Replication local replication cohort
• JHU replication cohort at Johns Hopkins University
• COL replication cohort at Columbia
• UT replication cohort at University of Utah
• FR-CRET replication cohort at Hopital Intercommunal de Creteil
• WASH-U replication cohort at Washington University
• Weight the effective sample size of each cohort if the ratio between cases and controls was equal to one based upon actual samples listed in Sl.
• Z-score weighted average and
• Odds Ratio weighted average odds ratio for the HDL-raising allele T compared to the major allele
• 95% CI confidence intervals for the odds ratio
• P-value F- value calculated from sum of weighted average Z score.
• the replication cohorts are (a) Tufts/MGH GWAS, (b) Tufts/MGH
• Serum Lipid Biomarkers and Hepatic Lipase Gene Associations with Age-Related Macular Degeneration.
• ABSTRACT Objective A genetic variant in the high density lipoprotein (HDL) cholesterol pathway, hepatic lipase (LIPC), was discovered to be associated with advanced age- related macular degeneration (AMD) in our genome-wide association study.
• AMD advanced age-related macular degeneration
• Hepatic lipase (LIPC), a gene located on chromosome 15q22, was recently discovered to be associated with age-related macular degeneration (AMD) in our large genome-wide association study (GWAS). 1 This finding was replicated in another GWAS. 2 This new variant encodes the hepatic lipase enzyme, and affects
• HDL-c serum high density lipoprotein cholesterol
• AREDS Age Related Eye Disease
• SNPs single nucleotide polymorphisms
• Complement Component 2 or C2 E318D (rs9332739), the non-synonymous coding SNP variant in exon 7 of C2 resulting in a substitution of aspartic acid for glutamic acid at codon 318, 5) Complement Factor B or CFB R32Q (rs641153), the non- synonymous coding SNP variant in exon 2 of CFB resulting in the substitution of
• Genotyping was performed using primer mass extension and MALDI-TOF MS analysis (MassEXTEND methodology of Sequenom, San Diego, CA) at the Broad 5 Institute Center for Genotyping and Analysis (Cambridge, MA).
• Smoking history was collected at onset of the study procedures from a standardized risk factor questionnaire. Smokers were defined as having smoked at 15 least one cigarette per day for six months or longer. Height and weight were measured at baseline to calculate body mass index (BMI) (weight in pounds multiplied by 703 divided by height in inches squared). Blood pressure was measured at the onset of the studies, and categorized as follows: 1) Normal: systolic less than 120 mm/Hg and diastolic less than 80 mm/Hg, 2) Prehypertension: systolic 20 120- 139 mm/Hg or diastolic 80-89 mm/Hg, 3) Hypertension Stage 1 : systolic 140-
• Hypertension Stage 2 systolic 160 mm/Hg or higher or diastolic 100 mm/Hg or higher.
• Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated for 25 genetic and behavioral variables using logistic regression controlling for age ( ⁇ ?9,
• BOST_1508298.1 values for each continuous lipid variable to evaluate whether lipids were related to LIPC genotype, controlling for age and sex.
• Model A was adjusted for age, sex, 5 smoking (never, past, current), BMI ( ⁇ 25, 25-29.9, 30+), and blood pressure (normal, pre-hypertension, hypertension stage 1 , hypertension stage 2).
• Model B was adjusted for the same covariates as in model A plus genotypes CFH Y402H(TT, CT, CC), CFH: rsl 410966 (TT, CT, CC), C2 (GG, CG/CC), CFB (CC, CTAT),
• Table 12 displays sex adjusted serum lipid means and ranges for controls, all cases, and the GA and NV groups separately. LDL was significantly higher for all
• Triglyceride level did not appear to be related to AMD in these analyses controlling for other variables.
• CVD Cardiovascular disease
• AMD have been shown to share some of the same risk factors including high BMI, C-reactive protein, other cytokines, and history of smoking. It is possible that CVD could provide a comparison model for the role of cholesterol in AMD pathogenesis, and several studies have evaluated the
• cholesterol also functions locally in the retina. Cholesterol has been found in drusen and in Bruch's membrane. LDL and HDL transport cholesterol, vitamin E, and lutein/zeaxanthin within the retinal pigment epithelium (RPE) and Bruch's membrane for use by photoreceptors. However, in older eyes these lipoproteins
• Hypertension has also been considered as a potential risk factor for AMD in
• LIPC is associated with reduced risk of GA and NV AMD.
• HDL may be associated with reduced risk of advanced AMD, and LDL and total cholesterol increased risk of AMD. Further investigation into the relationships among LIPC, serum lipids, and AMD as well as elucidation of other mechanisms involved in this new pathway is warranted.
• Hepatic lipase gene in the high-density lipoprotein cholesterol (HDL-c) pathway as a novel locus for AMD risk, with a protective effect for the minor T allele.
• Hepatic lipase is a form of lipase. It is expressed in the liver and
• LIPC encodes hepatic triglyceride lipase, which is expressed in the liver. LIPC has the dual functions of triglyceride hydrolase and ligand/bridging factor for receptor-mediated lipoprotein uptake. A separate GWAS independently corroborated the LIPC association with AMD but that finding did not reach the level
• the Age-Related Eye Disease Study included a randomized grouping of AREDS and randomized grouping of AREDS.
• DNA samples were obtained from the AREDS Genetic Repository.
• the single nucleotide polymorphism, rsl 0468017, a functional variant of the LIPC gene on chromosome 15q22 was assessed.
• variants in seven known AMD genes were also determined : l)the common single nucleotide polymorphism (SNP) in exon 9 of the
• Complement component 2 or C2 E318D (rs9332739), the non-synonymous coding SNP variant in exon 7 of C2 resulting in the amino acid glutamic acid changing to aspartic acid at codon 318, 5) Complement Factor B or CFB R32Q (rs641153), the non-synonymous coding SNP variant in exon 2 of CFB resulting in the amino acid
• the gene HTRAl adjacent to it may in fact be the AMD-susceptibility gene on
• BOST_1508298.1 rs 10490924 used in this study can be considered a surrogate for the causal variant which resides in this region.
• C2/CFB genes there are two independent associations to the C2/CFB locus, but because of linkage disequilibrium we do not know which of the two genes or both are functionally affected.
• Genotyping was 5 performed using primer mass extension and MALDI-TOF MS analysis by the MassEXTEND methodology of Sequenom (San Diego, CA) at the Broad Institute Center for Genotyping and Analysis, Cambridge, MA.
• Statistical Analyses Individuals with advanced AMD, as well as the GA and NV subtypes, were
• Multivariate unconditional logistic regression analyses were performed to evaluate the relationships between AMD and LIPC, controlling for age (70 or older, younger than 70); gender; education (high school or less, more than high school); cigarette smoking (never, past, current); BMI which was calculated as
• HDL is the major lipoprotein transporter of lutein and zeaxanthin. Changes in the efficiency of carotenoid delivery is one mechanism by which LIPC genetic variation could be related to AMD.
• the distribution of the demographic, personal, and lifestyle variables according to the LIPC genotypes for cases and controls with geographic atrophy and neovascular disease are shown in Table 16. There were no significant differences in 5 age, gender, education, smoking, BMI, AREDS treatment, or calorie- adjusted lutein among the LIPC genotypes.
• Table 17 displays the association between the LIPC gene and other known AMD genetic loci. There were no significant gene-gene interactions seen between this gene and the other loci.
• Table 18 shows the odds ratios for the multivariate models, comparing all advanced AMD cases, as well as GA and NV cases, with controls, for the LIPC variant, while adjusting for demographic and behavioral risk factors. Controlling for age, gender, education, smoking, body mass index, AREDS treatment, and dietary lutein in multivariate model 1 , advanced cases were less likely to have the T allele, with OR
• Table 18 also shows the associations between advanced AMD, GA, and NV with older age, less education, past and current smoking, high BMI, and lower levels of lutein, compared with controls.
• Cigarette smoking was associated with a statistically significant increased risk of advanced AMD for both subtypes, controlling for genotype and other factors,
• LIPC 15 behavioral factors including BMI, smoking, and dietary factors, as well as all known AMD genes.
• LIPC and environmental factors were independently associated with advanced AMD, the leading cause of visual impairment and vision-related reduced quality of life among elderly individuals. After adjustment for demographic and behavioral factors, the newly discovered variant of the LIPC gene located on
• Strengths of the study include the large, well characterized population of io Caucasian patients with and without advanced AMD from various geographic regions around the US, standardized collection of risk factor information, direct measurements of height and weight, and classification of maculopathy by ophthalmologic examinations and fundus photography. Misclassification was unlikely since grades were assigned without knowledge of risk factors or genotype.
• Inflammation and tissue remodeling occur in association with many chronic diseases, such as cancer, and genes that indicate a predisposition to cancer and autoimmune responses, and those involved in the tissue vascularization and metastatic properties of cancer cells are of particular interest. Genes that express proteins involved in tissue remodeling pathways are specifically noted as potential diagnostic markers of AMD.
• one embodiment of the invention includes polymorphisms in the genes encoding various metalloproteinases, such as TIMP3/SYn, SNP rs9621532 (SEQ ID NO:39), wherein the cytidine polymorphism is indicative of AMD or susceptibility to AMD.
• TIMP3/SYn various metalloproteinases
• SNP rs9621532 SEQ ID NO:39
• AMD in a similar manner as those lipid metabolism genes described above.
• a polynucleotide including SEQ ID No: 39 is used in hybridization assays, or the polymorphism is detected by antibody binding or mass spectroscopy, or by other gene sequence identification methods.
• a preferred embodiment includes the polynucleotide sequence affixed to an array or gene chip, more preferably in combination with other AMD predictive genes. The presence of the polymorphism is predictive of developing AMD or is predictive of progression of the disease. Lack of the polymorphism suggests lowered risk for AMD. In combination with the other factors described, computer models of patient risk can be generated.

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